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. 2012 Jul 12;160(1):450–463. doi: 10.1104/pp.112.198200

Figure 2.

Figure 2.

Implementation of O-glycosylation in Arabidopsis and tobacco BY-2 suspension cells. A, SDS-PAGE western-blot analysis of four Arabidopsis lines expressing either MUC1 (lanes 1 and 2) or MUC1-YFP (lanes 4 and 5) alone. The contrast of the image was adjusted to visualize very weak bands for MUC1 (lanes 1 and 2). Lane 3 shows results for the wild type (WT). B, SDS-PAGE western-blot analysis of two tobacco BY-2 suspension cell lines expressing MUC1-YFP alone (lane 1) and MUC1-YFP together with the 2A-linked O-glycosylation machinery T2-2A-CytoEpi (lane 2). The absence (−) or presence (+) of O-glycosylation machinery is indicated above the lanes. C, An Arabidopsis line transgenic for T7-tagged full-coding secreted IFNα2B expressed alone (−) or coexpressed with the O-glycosylation machinery CytoEpi-T2 (+). The glycosylation of His tag-purified IFNα2B was detected by VVA lectin-blot analysis. Total protein extracts from transgenic Arabidopsis leaves or BY-2 cell callus were loaded and blots reacted with MUC1-specific MAbs 5E10 and 5E5, where 5E5 is specific for GalNAc-glycosylated MUC1 (Tn-MUC1) and does not react with nonglycosylated MUC1. Approximately 30 μg of total protein was loaded in each lane.